Millions of women suffer from recurrent urinary tract infections (UTIs) every year despite current antimicrobial treatments. UTIs are usually caused by the Gram-negative bacterium Escherichia coIi. The iGram positive pathogen Enterococcus faecalis is also an important cause of UTIs, particularly in the inosocomial setting. This project will use a blend of genetics, high-resolution electron and immunoflourescent :microscopy, mutagenesis and well-established in vitro tissue culture and in vivo mouse cystitis models to gain an understanding of the bacterial factors which are involved in the molecular cross-talk between the host and the pathogen. This interaction determines whether an infection occurs and if the infection is quickly cleared with a return to sterility in the bladder or a persistent reservoir results. The genetic differences in sequence and regulation between K12 and clinical strains of E. coil (both symptomatic and ASB) will be determined, using sequencing and microarrays. These studies will provide the basis for both targeted and global mutagenesis analyses. The mechanisms by which UPEC proceeds through each step of pathogenesis will then be determined and the contributions of specific bacterial genes to virulence will be determined. The triggers for innate host defenses, specifically the interactions between bacterial LPS with the host TLR4 isignalling system will be analyzed. In addition, mutations that are developed in this Project, that are deficient in pathogenesis will be used in other projects to gain a detailed understanding of the host response and the molecular cross-talk that occurs as a consequence of host-pathogen interactions. These studies will provide insight into possible downstream sequelae, such as recurrent UTIs. The knowledge of general and !specific mechanisms of infection will be increased by investigating the molecular basis of how Enterococcus :interacts with the urinary tract and causes disease. The specific knowledge that will be gained in this project will: i. lead to a better understanding of the mechanisms by which bacteria causes acute, recurrent and chronic cystitis, ii. lead to improved methods of treating and preventing this ubiquitous disease and iii. establish a coordinated set of analyses and model systems which can be used to understand the fine molecular details of both the early and long-term consequences of the interaction between a pathogen and its host tissue. In addition, since a wide variety of diseases are caused by Gram-negative bacteria that infect or invade mucos surfaces, the knowledge gained will be applicable to many infectious d!seases.